I am processing a sample of a small membrane protein, that shows multiple conformations. I was wondering if someone has experience, and could tell me an optimal way to differentiate small differences in small membrane proteins. As currently my resolution is stuck between 4.0-4.5 A, I am wondering if it is still due to heterogeneity in my sample. I find that 3D Variability doesn’t work too well in my hands, and I usually work with ab-initio, heterogeneous refinement and just a tiny bit of 3D classification. But I am also almost certain that my settings are not entirely optimal.